Tobacco smoking is a risk factor for cancers of the GI (gastrointestinal) tract, including hepatocellular carcinoma that has seen increasing incidence in the United States. However, little is known about the tobacco constituents that are potentially responsible for the development of GI cancers in human smokers. Using a population sample, we reported that the heterocyclic aromatic amine, 2-amino-9H-pyrido[2,3-b]indole (AC), a rodent GI carcinogen, is present in the urine of tobacco smokers in a dose-dependent manner while nonsmokers are devoid of the compound in their urine. We hypothesize that high exposure to AC through smoking of cigarettes, and the propensity of AC to undergo bioactivation by enzymes expressed in the liver and the colorectum, provides a biochemical mechanism for the development of GI tract cancer in smokers. Our long-term goal is to determine if AC in tobacco smoke is causally related to GI tract cancer in smokers, and to elucidate the mechanistic pathways underlying this exposure-cancer relationship. The original 3 aims of the parent grant are: 1) to determine the extent of exposure to AC, by measuring the levels of AC in urine and stool of tobacco smokers participating in a smoking cessation study; 2) to evaluate the genotoxicity of AC in the colorectum of mice, by measurement of DNA adducts and aberrant crypt foci, recognized early biomarkers of neoplasia; and 3) to characterize the major pathways of AC metabolism in mice and humans, especially as they relate to DNA adduct formation at the target tissue. The above studies are essential in understanding the biochemical toxicology of AC in GI tract cancers. However, in order to definitively establish a causal link between AC exposure and cancer in human smokers using existing population-based cohorts with baseline biospecimens, biomarkers of AC that reflect the stable profile of exposure in subjects need to be in place. The objectives of this grant revision are: 1) to examine the reaction products of the carcinogenic metabolites of AC with serum albumin and hemoglobin, in order to develop AC adducts of these blood proteins as biomarkers, and 2) to develop analytical mass spectrometry methods for sensitive and precision measurements of AC-blood protein biomarkers, for future use in large-scale, population-based cohort studies. This proposed research is relevant to NIH's mission on public health and directly relates to assessing the safety of an over-looked carcinogen that exists in high levels in tobacco smoke. The proposed research will establish a set of biomarkers of AC reflecting a smoker's stable exposure profile that can be used in epidemiologic cohort studies to definitively link AC exposure to human cancers. The biomarkers also will provide a means to measure and compare the toxicity and carcinogenicity of different tobacco products. PUBLIC HEALTH RELEVANCE: 2-Amino-9H-pyrido[2,3-b]indole (AC) is the most abundant of the aromatic amine carcinogens formed in tobacco smoke. There is mounting evidence that it may be an important causal agent for the development of GI (gastrointestinal tract) tract cancers in smokers, including the rapidly fatal hepatocellular carcinoma that has seen increasing incidence in the United States during the past three decades. Characterization of the metabolic pathways involved in bioactivation and detoxication of AC in humans as they relate to GI tract cancers, in conjunction with the development and validation of long-lived biomarkers of AC for use in large scale, prospective cohort studies would serve to definitively establish the causal role between AC exposure and smoking-related GI tract cancers in humans.